Betacyanin Biosynthetic Genes and Enzymes Are Differentially Induced by (a)biotic Stress in <i>Amaranthus hypochondriacus</i>

<div><p>An analysis of key genes and enzymes of the betacyanin biosynthetic pathway in <i>Amaranthus hypochondriacus</i> (<i>Ah</i>) was performed. Complete cDNA sequence of <i>Ah</i> genes coding for cyclo-DOPA 5-O glucosyltransferase (<i>AhcDOPA5-GT</i>), two 4, 5-DOPA-extradiol-dioxygenase isoforms (<i>AhDODA-1</i> and <i>AhDODA-2</i>, respectively), and a betanidin 5-<i>O</i>-glucosyltransferase (<i>AhB5-GT</i>), plus the partial sequence of an orthologue of the cytochrome P-450 <i>R</i> gene (<i>CYP76AD1</i>) were obtained. With the exception <i>AhDODA-2</i>, which had a closer phylogenetic relationship to <i>DODA-like</i> genes in anthocyanin-synthesizing plants, all genes analyzed closely resembled those reported in related Caryophyllales species. The measurement of basal gene expression levels, in addition to the DOPA oxidase tyrosinase (DOT) activity, in different tissues of three <i>Ah</i> genotypes having contrasting pigmentation levels (green to red-purple) was determined. Additional analyses were performed in <i>Ah</i> plants subjected to salt and drought stress and to two different insect herbivory regimes. Basal pigmentation accumulation in leaves, stems and roots of betacyanic plants correlated with higher expression levels of <i>AhDODA-1</i> and <i>AhB5-GT</i>, whereas DOT activity levels coincided with pigment accumulation in stems and roots and with the acyanic nature of green plants, respectively, but not with pigmentation in leaves. Although the abiotic stress treatments tested produced changes in pigment levels in different tissues, pigment accumulation was the highest in leaves and stems of drought stressed betacyanic plants, respectively. However, tissue pigment accumulation in stressed <i>Ah</i> plants did not always correlate with betacyanin biosynthetic gene expression levels and/or DOT activity. This effect was tissue- and genotype-dependent, and further suggested that other unexamined factors were influencing pigment content in stressed <i>Ah</i>. The results obtained from the insect herbivory assays, particularly in acyanic plants, also support the proposal that these genes could have functions other than betacyanin biosynthesis.</p></div

Expression of betacyanin biosynthetic genes in response to water stress.

<p>Relative expression levels² were determined in leaves, stems and roots of <i>A. hypochondriacus</i> plants, with contrasting pigmentation patterns, subjected to water stress. Induced or repressed levels of expression (i.e. relative expression ≥1.5 or ≤0.5) are shown in bold text and italics, respectively.</p>1<p>The genotypes examined in this study were <i>Ah</i> cv. Nutrisol (<i>AhNut</i>; with predominantly betacyanic leaves), <i>Ah</i> India Red (<i>AhIR</i>; with predominantly betacyanic stems) and <i>Ah</i> India Green (<i>AhIG</i>; with all tissues acyanic).</p>2<p>The fold change in the expression of the target genes was calculated using the 2^−ΔΔCt method according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099012#pone.0099012-Livak1" target="_blank">[76]</a>.</p

Expression of betacyanin biosynthetic genes in response to <i>continuous</i> insect herbivory.

<p>Relative expression levels³ were determined in stems of <i>A. hypochondriacus</i> plants, with contrasting pigmentation patterns, subjected to insect herbivory. Induced or repressed levels of expression (i.e. relative expression ≥1.5 or ≤0.5) are shown in bold text and italics, respectively.</p>1<p>h =  time, in hours, when the tissues were sampled after an 18 h period of continuous insect feeding.</p>2<p>The genotypes examined in this study were <i>Ah</i> cv. Nutrisol (<i>AhNut</i>; with predominantly betacyanic leaves), <i>Ah</i> India Red (<i>AhIR</i>; with predominantly betacyanic stems) and <i>Ah</i> India Green (<i>AhIG</i>; with all tissues acyanic).</p>3<p>The fold change in the expression of the target genes was calculated using the 2^−ΔΔCt method according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099012#pone.0099012-Livak1" target="_blank">[76]</a>.</p

Changes in pigment levels and tyrosinase activity in amaranth plants subjected to <i>continuous</i> insect herbivory.

<p>Amaranthine (<b>A</b>) and DOPA oxidase tyrosinase (DOT) activity (<b>B</b>) levels measured in leaves, stems and roots of plants of <i>A. hypochondriacus</i> genotypes (<i>AhNut</i> [N], <i>AhIR</i> [R] and <i>AhIG</i> [G]) having different patterns of pigmentation, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099012#pone-0099012-g001" target="_blank">Figure 1</a>, and subjected to <i>continuous</i> insect herbivory for 18 h and then sampled at 6, 24 and 72 h after larval removal. Mean values ± SE in control (C: empty bars) and treated (T: colored bars) plants are presented (n = 6). Asterisks over the bars represent statistically different values at <i>P</i>≤0.05 (Dunnetts test). Experiments were performed twice, and representative results are shown. FW  =  fresh weight.</p

Expression of betacyanin biosynthetic genes in response to <i>discontinuous</i> insect herbivory.

<p>Relative expression levels³ were determined in stems of <i>A. hypochondriacus</i> plants, with contrasting pigmentation patterns, subjected to insect herbivory. Induced or repressed levels of expression (i.e. relative expression ≥1.5 or ≤0.5) are shown in bold text and italics, respectively.</p>1<p>h =  time, in hours, spent on the plant by the feeding larvae before they were removed and the tissues sampled.</p>2<p>The genotypes examined in this study were <i>Ah</i> cv. Nutrisol (<i>AhNut</i>; with predominantly betacyanic leaves), <i>Ah</i> India Red (<i>AhIR</i>; with predominantly betacyanic stems) and <i>Ah</i> India Green (<i>AhIG</i>; with all tissues acyanic).</p>3<p>The fold change in the expression of the target genes was calculated using the 2^−ΔΔCt method according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099012#pone.0099012-Livak1" target="_blank">[76]</a>.</p

Changes in pigment levels and tyrosinase activity in water-stressed amaranth plants with contrasting pigmentation patterns.

<p>Amaranthine <b>(A)</b> and DOPA oxidase tyrosinase (DOT) activity <b>(B)</b> levels measured in leaves, stems and roots of water-stressed plants of <i>A. hypochondriacus</i> genotypes (<i>AhNut</i> [N], <i>AhIR</i> [R] and <i>AhIG</i> [G]) having different patterns of pigmentation, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099012#pone-0099012-g001" target="_blank">Figure 1</a>. Mean values ± SE in control (C; empty bars) and treated (colored bars) are presented (n = 6). Asterisks over the bars represent statistically different values at <i>P</i>≤0.05 (Tukey Kramer test). Experiments were performed twice, and representative results are shown. FW  =  fresh weight.</p

Expression of betacyanin biosynthetic genes in response to salt stress.

<p>Relative expression levels² were determined in leaves, stems and roots of <i>A. hypochondriacus</i> plants, with contrasting pigmentation patterns, subjected to salt stress. Induced or repressed levels of expression (i.e. relative expression ≥1.5 or ≤0.5) are shown in bold text and italics, respectively.</p>1<p>The genotypes examined in this study were <i>Ah</i> cv. Nutrisol (<i>AhNut</i>; with predominantly betacyanic leaves), <i>Ah</i> India Red (<i>AhIR</i>; with predominantly betacyanic stems) and <i>Ah</i> India Green (<i>AhIG</i>; with all tissues acyanic).</p>2<p>The fold change in the expression of the target genes was calculated using the 2^−ΔΔCt method according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099012#pone.0099012-Livak1" target="_blank">[76]</a>.</p

Pigment levels and tyrosinase activity in <i>A. hypochondriacus</i> plants with contrasting pigmentation patterns.

<p>Basal amaranthine (<b>A</b>) and DOPA oxidase tyrosinase activity (DOT) (<b>B</b>) levels measured in leaves, stems and roots of plants of <i>A. hypochondriacus</i> genotypes having different patterns of pigmentation: <i>AhNut</i> (predominantly betacyanic leaves, [N]; blue bars); <i>AhIR</i> (green leaves with pigmented vasculature and betacyanic stems, [R]; red bars) and <i>AhIG</i> (all tissues acyanic, [G]; green bars). Mean values ± SE are presented (n = 6). Different letters over the bars represent statistically different values at <i>P</i>≤0.05 (Tukey Kramer test). FW  =  fresh weight.</p

Changes in pigment levels and tyrosinase activity in salt-stressed amaranth plants with contrasting pigmentation patterns.

<p>Amaranthine <b>(A)</b> and DOPA oxidase tyrosinase (DOT) activity <b>(B)</b> levels measured in leaves, stems and roots of salt-stressed plants of <i>A. hypochondriacus</i> genotypes (<i>AhNut</i> [N], <i>AhIR</i> [R] and <i>AhIG</i> [G]) having different patterns of pigmentation, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099012#pone-0099012-g001" target="_blank">Figure 1</a>. Mean values ± SE in control (C; empty bars) and treated (colored bars) are presented (n = 6). Asterisks over the bars represent statistically different values at <i>P</i>≤0.05 (Tukey Kramer test). Experiments were performed twice, and representative results are shown. FW  =  fresh weight.</p

Changes in pigment levels and tyrosinase activity in amaranth plants subjected to <i>discontinuous</i> insect herbivory.

<p>Amaranthine (<b>A</b>) and DOPA oxidase tyrosinase (DOT) activity (<b>B</b>) levels measured in leaves, stems and roots of plants of <i>A. hypochondriacus</i> genotypes (<i>AhNut</i> [N], <i>AhIR</i> [R] and <i>AhIG</i> [G]) having different patterns of pigmentation, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099012#pone-0099012-g001" target="_blank">Figure 1</a>, and subjected to <i>discontinuous</i> (samples were taken on removing the larvae) insect herbivory for 3, 9 and 15 h. Mean values ± SE in control (C: empty bars) and treated (colored bars) plants are presented (n = 6). Asterisks over the bars represent statistically different values at <i>P</i>≤0.05 (Dunnetts test). Experiments were performed twice, and representative results are shown. FW  =  fresh weight.</p